Many systems have been described for the expression of nucleic acids encoding e.g., polypeptides such as recombinant proteins in prokaryotic systems. The T7 RNA polymerase (T7RNAP, SEQ ID NO:17) based expression systems are the most widely used systems to produce recombinant proteins in Escherichia coli (Studier, et al. (1990) Methods Enzymol 185:60-89). It is of note that besides in E. coli, T7RNAP based expression has been used in a host of different pro- and eukaryotic organisms (e.g., Kang et al. (2007) Protein Expr Purif. 55(2):325-33; Dower and Robasch (2002) RNA 8:686-697; Nguyen et al. (2004) Plant Biotechnol. J. 2: 301-310; Polkinghorne and Roy (1995) Nucleic Acids Res. 23(1): 188-191; Wang et al. (2007) Analytical Biochemistry, in press (doi:10.1016/j.ab.2007.11.037)). T7RNAP recognizes a very specific promoter, i.e., a T7 promoter (Chamberlin et al., (1970) Nature 228, 227-231; Oakley and Coleman (1977) PNAS 74, 4266-4270; Rosa (1979) Cell 16, 815-825;
Panayotatos and Wells (1979) Nuci. Acids Res. 13, 2227-2240; Dunn and Studier (1983) J. Mol. Biol. 166, 477-535/175, 111-112), and transcribes DNA with an eight times higher transcription rate than E. coli RNA polymerase (Iost, et al. (1992) J Bacteriol 174:619-22). This is of great advantage for the high yield production of recombinant protein in E. coli. The down-side of good efficiency is e.g., the large metabolic burden and folding stress imposed onto the host cell. While the production of proteins in the cytoplasm of E. coli is relatively straightforward, overexpression of membrane proteins in E. coli remains a challenging task (Wagner, et al. (2006) Trends Biotechnol 24:364-71). Although membrane proteins can often easily be expressed in inclusion bodies, their refolding into functional proteins is usually not successful. Overexpression of membrane proteins through accumulation in the cytoplasmic membrane system avoids this refolding problem, but is usually toxic, thereby severely reducing yields. This is primarily caused by the complex requirements of membrane protein biogenesis where, after translation initiation at the ribosome, membrane protein ribosome nascent chain complexes get targeted to the cytoplasmic membrane (Luirink, et al. (2005) Annu Rev Microbiol 59:329-55). Transmembrane domains (TMDs) of membrane proteins get trapped in the Sec translocon and subsequently partition into the lipid bilayer. Membrane protein overexpression easily saturates one or several steps of the biogenesis pathway which leads to undesirable aggregation and degradation of recombinant protein in the cytoplasm. Furthermore, blockage of the secretory pathway results in severe toxicity for the host cell (Wagner, et al. (2007) Mol Cell Proteomics 6:1527-50). Reduced viability of the host cell and misfolding of the overexpressed recombinant protein result in low yields of the desired product. The major problem in this case is that expression of polypeptides by T7RNAP is too strong. Most T7RNAP based expression systems have relied exclusively on a fixed activity of T7RNAP resulting in a fixed intensity of recombinant protein expression. The most widely used strains for this purpose are BL21(DE3) and its derivatives BL21(DE3)pLysS, BL21(DE3)pLysE, C41(DE3) and C43(DE3) (Studier (1991) J Mol Biol 219:37-44) (Miroux (1996) J Mol Biol 260: 289-98). Hosts carrying the pLysS and pLysE plasmids express T7 lysozyme, pLysS at a set low level and pLysE at a set high level. All of these fixed systems leave no opportunity to adjust the intensity of expression to the individual requirements of different target proteins. Since it is unpredictable what expression intensity is optimal for getting the best overexpression yields of a particular protein, expression has to be screened in different strains with fixed T7RNAP based expression intensities. It would be ideal if different expression intensities could be screened for in only one T7RNAP based expression system. Such “all in one” expression system would facilitate overexpression screening tremendously. Thus, there is a need to provide alternative prokaryotic and eukaryotic expression systems with the ability to continuously adjust the expression intensity of nucleic acid sequences, particularly those encoding membrane proteins and other polypeptides with complex post-translational biogenetic requirements; e.g., secretory proteins, proteins that are post-translationally modified (e.g., glycosylated (Wacker et al., (2002) Science 298, 1790 -1793), and proteins that require assistance of chaperones for their folding.
Finding the optimal T7RNAP based strain for the overexpression of a protein is a matter of time consuming ‘trial and error’, whereby e.g., BL21 (DE3), BL21 (DE3)pLysS, BL21 (DE3)pLysS (Studier (1991) J Mol Biol 219: 37-44), C41 (DE3) and C43 (DE3) are used (Miroux (1996) J Mol Biol 260, 289-98). In these strains, T7RNAP activity is fixed and the most suitable strain must be screened for overexpression of the desired protein. What strain will be best is something one cannot predict.